Antibodies are made up of two classes of polypeptide chains, light chains and heavy chains. A single naturally occurring antibody comprises two identical copies of a light chain and two identical copies of a heavy chain. The heavy chains, which each contain one variable domain and multiple constant domains, bind to one another via disulfide bonding within their constant domains to form the “stem” of the antibody. The light chains, which contain one variable domain and one constant domain, each bind to one heavy chain via disulfide binding. The variable domain of each light chain is aligned with the variable domain of the heavy chain to which it is bound. The variable regions of both the light chains and heavy chains contain three hypervariable regions sandwiched between four more conserved framework regions. These hypervariable regions, known as the complementary determining regions (CDRs), form loops that comprise the principle antigen binding surface of the antibody.
Monoclonal antibodies are antibodies that are derived from a single source or clone of cells that recognize only one antigen epitope. Generally, they are made by fusing an immortalized tumor cell with a mammalian immune cell to form a hybridoma cell that produces an antibody. This hybridoma cell is capable of producing a large quantity of a single antibody. The production of monoclonal antibodies is generally done using rat or mouse cells, but other species such as hamsters, sheep, and humans have been used. Monoclonal antibodies possess a variety of potential in vivo uses. For instance, labeled monoclonal antibodies that specifically recognize a particular tumor-associated antigen can be an extremely powerful diagnostic tool. One key issue for the in vivo therapeutic use of monoclonal antibodies has been the response of the human immune system to xenogeneic antibodies. Clinical studies with murine monoclonal antibodies have shown effective tumor targeting, but have also resulted in rapid clearance of the murine antibody due to the generation of a human anti-murine antibody (HAMA) immune response (Schroff 1985; Shawler 1985).
One solution to HAMA response problem is to generate human antibodies from human immunoglobulin phage display libraries (Winter 1994) or transgenic animals (Bruggemann 1991, Mendez 1997). These techniques have produced a small yet growing number of antibodies with high specificity and affinity. However, antibodies produced by these methods have either exhibited specificity only for immobilized antigen or have exhibited poor expression as intact antibodies in mammalian cell culture. The question remains to be answered in the clinic whether this new generation of engineered antibodies will be immunogenic, if not through a response to the foreign framework residues then as an anti-idiotypic response. Another solution to the HAMA response problem has been the use of recombinant methodologies to generate chimeric monoclonal antibodies, which generally consist of a murine antigen-binding variable domain coupled to a human constant domain. These chimeras have a lower frequency of immune response, but they are not effective for all antibodies and may still generate an immune response against the murine variable region. A third solution to the HAMA response problem is the utilization of humanized or reshaped monoclonal antibodies. These consist of human antibodies in which only the complementary determining region (CDR) has been substituted with an animal CDR region.
The current generation of humanized monoclonal antibodies approved for therapy are the result of grafting murine-derived CDR's onto a human antibody framework (Jones 1986; Low 1986). This process of CDR-grafting is a well established technique, but it has a downside in that it frequently generates an antibody with substantially decreased antigen binding affinity compared to the parental antibody. This decreased affinity is the result of unanticipated steric clashes between the human immunoglobulin framework and the murine CDR side chains, which alters the CDR loop conformation. This disadvantage can be overcome by the reiterative process of back-mutagenesis, which involves the restoration of key murine framework residues that are responsible for maintaining the correct CDR loop formations (Foote 1992). However, this process is laborious and random.
T84.66 is a murine monoclonal antibody with high specificity and affinity for carcinoembryonic antigen (CEA). CEA is one of the most well characterized human tumor-associated antigens (Wagener 1983). It is a glycoprotein that has limited expression in normal adults, and is commonly overexpressed in carcinomas of the colon, rectum, breast, lung, pancreas, and gastrointestinal tract (Marshall 2003). In fact, CEA is expressed on nearly 50% of all human tumors (Huang 2002). Increased CEA expression promotes intercellular adhesions, which may lead to metastasis (Marshall 2003).
T84.66 has an extensive clinical history, and has been used in the radioimmunotherapy treatment of over 200 patients. Radiolabeled murine T84.66 monoclonal antibody evaluated in the clinic is capable of imaging 69% of primary colorectal carcinomas prior to surgery (Beatty 1986), but it also generates a HAMA response (Morton 1988). The genes for the T84.66 antibody were cloned and a human-murine chimeric version (cT84.66) was expressed in mammalian cells (Neumaier 1990). In a pilot imaging study for colorectal disease using a single administration, only one out of 29 patients exhibited a human anti-chimeric antibody (HACA) response against cT84.66 (Wong 1997). However, as multiple administration immunotherapy trials have proceeded (Wong 1995; Wong 1999), an increase in the frequency of the HACA response has been noted. Thus, there is a need in the art for humanized T84.66 antibodies that maintain the high specificity and affinity of the parental antibody while minimizing the HAMA response.